It is thought that the initial step in the pathway leading to carcinogenesis is damage to DNA. Cells have developed the capability to cope with damage to their DNA through the expression of a series of specific DNA repair enzymes. The long term goal of my research is (1) to have a better understanding of the biochemical properties of these DNA repair enzymes and their role in the induction of mutagenesis and ultimately carcinogenesis; (2) to determine the role of ionizing radiation as a DNA damaging agent and carcinogen; (3) to have a better understanding of human diseases associated with a defect in DNA repair such as ataxia telangiectasia. To begin to accomplish part of these long term objectives, I have chosen two areas of concentration. The first is the characterization of the novel DNA repair enzyme DNA deoxyribophosphodiesterase, or dRpase. The enzyme, initially isolated from E. coli, catalyzes the release of 2-deoxyribose-5-phosphate from single- strand interruptions in DNA with a base-free residue on the 5' side. To further characterize this repair activity, it is proposed (1) to determine the importance of he dRpase activity in the DNA base excision repair pathway for the removal of altered bases and apurinic sites by measuring the incorporation of radiolabelled nucleoside triphosphates into defined oligonucleotide and plasmid substrates in vitro; (2) to determine the substrate specificity of the dRpase activity through the use of specific oligonucleotide and plasmid substrates containing modified sugar residues; (3) to clone the gene coding for the dRpase activity by (a) overproduction of the activity in E. coli, or (b) through further purification of the protein to allow for the production of a mixed oligonucleotide to screen a genomic library containing the dRpase gene. The cloned dRpase gene will be used to overproduce the enzyme in E. coli to obtain large amounts for biochemical studies, and the DNA sequence information will be used to determine the molecular organization of the gene and its relation to other known DNA repair genes. The second area of focus is to gain a better understanding of the biochemical basis for the human inherited disease ataxia telangiectasia (A-T). It has been recently discovered that lysates of cells derived from A-T patients contain an activity which modifies DNA specifically at deoxyribose-phosphate moieties present at DNA strand breaks. To further understand the biochemical events occurring in the A-T cell lysates, it is proposed to determine the exact chemical structure of the modified deoxyribose-phosphate product produced in the A-T cell lysates and to purify the enzymatic activities present in the A-T cell lines responsible for the modification.